Peptide fragments for inducing synthesis of extracellular matrix proteins

ABSTRACT

Short biologically active tetrapeptides are disclosed that are comprised of the sequences GxxG and PxxP where G (glycine) and P (proline) are maintained and x is a variable amino acid. The peptides can be used singly or in combination to stimulate production of extracellular matrix proteins in skin. A rapid, low-cost method of producing heterogenous formulations of tetrapeptides is disclosed.

This application claims the benefit of priority to U.S. ProvisionalApplication Ser. No. 60/813,284, filed Jun. 13, 2006, which is hereinincorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to tetrapeptides with the amino acid motif GxxG orPxxP, where G (glycine) and P (proline) are maintained and x is avariable amino acid. The invention also relates to frame shift activetetrapeptides which are tetrapeptide sequences shifted one frame from aGxxG or PxxP tetrapeptide in an ECM protein. In particular, theinvention relates to GxxG, PxxP, or frame shift active peptides thatstimulate production of extracellular matrix proteins and enhance woundclosure of the epithelial cell monolayer of scratch-wounded human skin.The peptide compositions may be used in formulations for repairingdamaged skin or maintaining healthy skin.

BACKGROUND OF THE INVENTION

Skin aging is commonly viewed as wrinkle formation and impaired woundhealing. A wound is defined as a break in the epithelial integrity ofthe skin. Normal wound healing involves a complex and dynamic butsuperbly orchestrated series of events leading to the repair of injuredtissues. The largest component of normal skin is the extracellularmatrix (ECM), a gel-like matrix produced by the cells that it surrounds.The ECM is composed of two major classes including fibrous structuralproteins and proteoglycans. Changes in the composition and crosslinkedstate of the ECM are known to be associated with aging and a range ofacquired and heritable skin disorders. It has been well documented thatECM not only provides structural support, but also influences cellularbehavior such as differentiation and proliferation. Also, more and moreresearch suggests that the matrix components may be a source of cellsignals to facilitate epithelial cell proliferation and migration andthus enhance wound healing.

The largest class of fibrous ECM molecules is the collagen family, whichincludes at least 16 different types of collagen. Collagen in the dermalmatrix is composed primarily of type I (80-85%) and type III (8-11%)collagens, both of which are fibrillar, or rod-shaped, collagens. Thetensile strength of skin is due predominately to these fibrillarcollagen molecules, which self-assemble into microfibrils in ahead-to-tail and staggered side-to-side lateral arrangement. Collagenmolecules become cross-linked to adjacent collagen molecules, creatingadditional strength and stability in collagen fibers. Damage to thecollagen network (e.g. by enzymes or physical destruction), or its totalcollapse causes healing to take place by repair.

Various bioactive peptides that stimulate production of ECM proteinshave been reported in both the scientific literature and in issuedpatents. Peptides historically have been isolated from natural sourcesand have recently been the subject of structure-function relationshipstudies. Natural peptides have also served as starting points for thedesign of synthetic peptide analogs.

Specific sequences within ECM proteins can stimulate useful elements inskin, such as type I collagen, type III collagen, and fibronectin(Katayama et. al., J. BIOL. CHEM. 288: 9941-9944 (1983)). Katayama etal. identified the pentapeptide, KTTKS (SEQ ID NO:17), within thecarboxy-terminal propeptide (residues 197-241) of type I collagen. Thepropeptide is cleaved during production of the mature collagen protein.The cleaved propeptide may participate in regulating collagen productionvia a biosynthesis feedback mechanism, with the KTTKS segment playing anactive role. Maquart et al. (J Soc BIOL. 193: 423-28 (1999)) reportedthat the peptides GHK and CNYYSNS also stimulate ECM synthesis. Thesesequences may be released during ECM turnover, thereby signaling theneed for ECM repair. The short peptide sequences liberated by eithermechanism are often called “matrikines” (Maquart et al., J. Soc. BIOL.193: 423-28 (1999)).

While a number of natural and synthetic peptides exist, there is a needfor improved biologically active peptides and methods for their use.

SUMMARY OF THE INVENTION

Tetrapeptides are disclosed that are characterized by the amino acidsequence motif GxxG or PxxP, where G (glycine) and P (proline) residuesare maintained and x is a variable amino acid. The tetrapeptides arederived from sequences that occur multiple times throughout the primarysequence of the ECM protein, type IV collagen. The disclosed sequencesinduce production of all forms of collagen more than previously knownpeptide sequences, including KTTKS, sold under the trademark MATRIXYL™by SEDERMA SAS (France). Further, a composition comprising a combinationof various multiply-repeating sequences elicits an even greatercollagen-producing response. Additional benefits may be expected frompeptide combinations present in a variety of ECM proteins.

Producing a specific combination of tetrapeptides for ECM rebuilding canbe commercially cost-prohibitive. A relatively simple and cost-effectivemeans of producing a diverse combination of biologically activetetrapeptides is disclosed. By producing a combinatorial library oftetrapeptides with the GxxG or PxxP motif, a variety of biologicallyactive tetrapeptides can be generated in the same manufacturing run(e.g., GEPG, GPEG, GPPG, and GEEG). The combination of tetrapeptides mayinduce more formation of ECM proteins than single peptides. Compositionscomprising the disclosed tetrapeptides, alone or in combination, areuseful in skin care markets including, but not limited to, those thataddress skin wrinkling, toning, firmness, or sagging. The stimulation ofcollagen by the disclosed tetrapeptides can significantly improve thehealth and appearance of damaged and aged skin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is SEQ ID NO:45 which is the Collagen IV amino acid sequenceillustrating the occurrences of GxxG tetrapeptides. All bold sequencesare underlined and overlapping sequences are double-underlined.

FIG. 2 is SEQ ID NO:46 which is the Collagen III amino acid sequenceillustrating the occurrences of the frame shift actives PGPR and GAGP.All frame shift active sequences are bold and underlined and the GxxGsequences occurring one frame shift away are double-underlined.

FIG. 3 is also SEQ ID NO:45, the Collagen IV amino acid sequence,illustrating the occurrences of the tetrapeptide PGPP.

DETAILED DESCRIPTION OF THE INVENTION

The invention is generally directed towards tetrapeptides that stimulateproduction of ECM proteins and modulate wound healing, and uses of suchtetrapeptides.

Peptides

One embodiment of the invention is directed towards an isolatedtetrapeptide comprising the motif GxxG or PxxP. In this embodiment G(glycine) or P (proline) is maintained and x is a variable amino acid.The peptide can generally be any peptide that falls within the abovedescription, and more preferably is SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12. SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, orSEQ ID NO:16.

Another embodiment of the invention is directed towards an isolatedtetrapeptide comprising the motif GxPG, where x is P at either variableposition, or both. In this embodiment, G (glycine) and P (proline) aremaintained and x is a variable amino acid. The peptide can generally beany peptide that falls within the above description, and more preferablyis SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7.

Another embodiment of the invention is directed towards an isolatedtetrapeptide comprising the motif GExG. In this embodiment, G (glycine)and E (glutamic acid) are maintained and x is a variable amino acid. Thepeptide can generally be any peptide that falls within the abovedescription, and more preferably is SEQ ID NO:5 or SEQ ID NO:8.

Another embodiment of the invention is directed towards an isolatedtetrapeptide comprising the motif PGxP. In this embodiment, P (proline)and G (glycine) are maintained and x is a variable amino acid. Thepeptide can generally be any peptide that falls within the abovedescription, and more preferably is SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:14, or SEQ ID NO:16.

Another embodiment of the invention is directed towards an isolatedtetrapeptide comprising the motif PExP. In this embodiment, P (proline)and E (glutamic acid) are maintained and x is a variable amino acid. Thepeptide can generally be any peptide that falls within the abovedescription, and more preferably is SEQ ID NO:1 or SEQ ID NO:9.

Another embodiment of the invention is directed towards a frame shiftactive tetrapeptide. In this embodiment, the tetrapeptide occurs oneframe shift from either a GxxG or PxxP tetrapeptide in an ECM protein.The peptide can generally be any peptide that falls within the abovedescription, and more preferably is SEQ ID NO:4 or SEQ ID NO:6.

Each of the above-described peptides can comprise D- or L-amino acids.The peptides can comprise all D-amino acids or L-amino acids. Thepeptides can have an acid C-terminus (—CO₂H) or, preferably, an amideC-terminus (—CONH₂, —CONHR, or —CONR₂). The peptides may be furtheraugmented or modified, either chemically or enzymatically. For example,the peptides may be amidated (—NH₂) on the C-terminus, which may renderthe tetrapeptide less susceptible to protease degradation and increasetheir solubility compared to the free acid forms. The peptides may alsobe lipidated which may provide for enhanced skin penetration.

The above-described peptides may contain the following amino acids: R(arginine), L (leucine), P (proline), F (phenylalanine), Q (glutamine),E (glutamic acid), I (isoleucine), K (lysine), S (serine), V (valine), A(alanine), N (asparagine), D (aspartic acid), T (threonine), Y(tyrosine) and G (glycine). The above-described peptides do not includethe following M (methionine), C (cysteine), H (histidine) or W(tryptophan). Accordingly, in one embodiment, x is not selected fromeither (methionine), C (cysteine), H (histidine) or W (tryptophan).

Methods of Use

An additional embodiment of the invention is directed towards methods ofusing the above-described peptides. The methods of use may involve theuse of a single peptide, or may involve the use of two or more peptidesin combination.

An embodiment of the invention is a method of promoting repair ofdamaged skin and maintenance of healthy skin using tetrapeptides thatstimulate production of ECM proteins. The method generally is directedtowards contacting dermal (skin) cells with a composition containing thepeptide. The compositions can be an aerosol, emulsion, liquid, lotion,cream, paste, ointment, foam, or other pharmaceutically acceptableformulation. Generally, a pharmaceutically acceptable formulation wouldinclude any acceptable carrier suitable for use on human skin, e.g.cosmetically acceptable carrier and dermatological acceptable carrier.The compositions may contain other biologically active agents such asretinoids or other peptides. The compositions may containpharmaceutically acceptable carriers or adjuvants. The contacting stepcan be performed in vivo, in situ, in vitro, or by any method known tothose of skill in the art. Most preferably, the contacting step is to beperformed topically at a concentration sufficient to elicit astimulatory response. The concentration of the peptide in thecomposition can be about 0.01 μg/mL to about 100 1μg/mL, about 0.1 μg/mLto about 50 μg/mL, and about 0.1 μg/mL to about 1 μg/mL. The contactingstep can be performed on a mammal, a cat, a dog, a cow, a horse, a pig,or a human. A preferred composition for promoting ECM protein productioncomprises SEQ ID NO:8; more preferably, the composition comprises SEQ IDNO:8 in a heterogeneous mixture with at least one other tetrapeptide. Ina most preferred embodiment, the individual tetrapeptides in thecomposition would cause sustained collagen production over a period ofat least 48 hours.

An additional embodiment of the invention is directed towards a methodfor promoting wound healing of skin damaged by normal aging, disease,injury, trauma, or by surgery or other medical procedures. The methodcan comprise administering to the wound of an animal a composition,wherein the composition comprises any of the above-described peptides,singularly or in combination. The compositions can be a liquid, lotion,cream, paste, ointment, foam, or any other pharmaceutically acceptableformulation. The compositions may contain pharmaceutically acceptablecarriers or adjuvants. The compositions may contain other biologicallyactive agents such as antimicrobial agents or growth factors. Thecompositions may also be used in combination with other therapeuticagents such as tissue grafts, tissue culture products, oxygen ordressings. The concentration of the peptide in the composition can beabout 0.01 μg/mL to about 100 μg/mL, about 0.1 μg/mL to about 50 μg/mL,and about 0.1 μg/mL to about 1 μg/mL. The composition can beadministered to the wound topically. The animal can generally be anykind of animal, and preferably is a mammal, and more preferably is ahuman, cow, horse, cat, dog, pig, goat, or sheep. A preferredcomposition for wound healing applications in which ECM proteinproduction is promoted comprises SEQ ID NO:8; more preferably, thecomposition comprises SEQ ID NO:8 in a heterogeneous mixture with atleast one other tetrapeptide. In a most preferred embodiment, theindividual tetrapeptides in the composition would cause sustainedcollagen production over a period of at least 48 hours.

An additional embodiment of the invention is directed towards a methodfor reducing scarring of skin damaged by normal aging, disease, injury,trauma, or by surgery or other medical procedures. The method cancomprise administering to the wound of an animal a composition, whereinthe composition comprises any of the above-described peptides,singularly or in combination. The compositions can be a liquid, lotion,cream, paste, ointment, foam, or other pharmaceutically acceptableformulation. The compositions may contain pharmaceutically acceptablecarriers or adjuvants. The compositions may contain other biologicallyactive agents such as antimicrobial agents or growth factors. Thecompositions may also be used in combination with other therapeuticagents such as tissue grafts, tissue culture products, oxygen ordressings. The concentration of the peptide in the composition can beabout 0.01 μg/mL to about 100 μg/mL, about 0.1 μg/mL to about 50 μg/mL,and about 0.1 μg/mL to about 1 μg/mL. The composition can beadministered to the wound topically. The animal can generally be anykind of animal, and preferably is a mammal, and more preferably is ahuman, cow, horse, cat, dog, pig, goat, or sheep. A preferredcomposition for wound healing applications in which ECM proteinproduction is promoted comprises SEQ ID NO:8; more preferably, thecomposition comprises SEQ ID NO:8 in a heterogeneous mixture with atleast one other tetrapeptide. In a most preferred embodiment, theindividual tetrapeptides in the composition would cause sustainedcollagen production over a period of at least 48 hours.

A further embodiment of the invention is directed towards a method forproducing the disclosed tetrapeptides in combination. The peptides maybe produced using any method known to those skilled in the art such asthose disclosed in Merrifield, R. B., Solid Phase Peptide Synthesis I,J. AM. CHEM. Soc. 85: 2149-2154 (1963); Carpino, L. A. et al.,[(9-Fluorenylmethyl)Oxy] Carbonyl (Fmoc) Amino Acid Chlorides:Synthesis, Characterization, And Application To The Rapid Synthesis OfShort Peptides, J. ORG. CHEM. 37: 51: 3732-3734; Merrifield, R. B. etal., Instrument For Automated Synthesis Of Peptides, ANAL. CHEM. 38:1905-1914 (1966); or Kent, S. B. H. et al., High Yield ChemicalSynthesis Of Biologically Active Peptides On An Automated PeptideSynthesizer Of Novel Design, IN: PEPTIDES 1984 (Ragnarsson U., ed.)Almqvist and Wiksell Int., Stockholm (Sweden), pp. 185-188, all of whichare incorporated by reference herein in their entirety. Preferably, thepeptides will be produced by a machine capable of sequential addition ofamino acids to a growing peptide chain. However, the peptides may alsobe manufactured using standard solution phase methodology.

It has been observed that the addition of a mixture of free amino acidsinstead of homogenous peptide mixtures during peptide chain synthesisresults in varied incorporation of free amino acids such that acombination of peptides results from the synthesis reactions. Therelative incorporation frequency of a particular amino acid included ina mixture of two or more amino acids added during synthesis may beadjusted. Adjustment is made possible by modifying the ratio of a freeamino acid made available during the synthesis process relative to theother amino acids in the mixture (this is termed an isokinetic mixture).

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLES Example 1 Identification of Repeat Tetrapeptide Sequences inCollagen

A relatively high proportion of collagen IV tetrapeptide repeatsequences have the motif GxxG (where x is any amino acid). A number ofthese are shown in situ as part of the full collagen IV sequenceillustrated in FIG. 1 as SEQ ID NO:45. Collagen IV was examined firstdue to its role of interacting with other specialized ECM components(See Gregory Schultz et al., 2005). There are eleven sequences with theGxxG motif in collagen IV that appear more than ten times (GxxG where xxis represented by: vp, ek, fp, lp, pp, sp, ep, ip, pk, qp and tp). Ofthese tetrapeptide sequences, eight of eleven sequences contain prolinein position 3, two of eleven sequences contain P in position 2, one ofeleven sequences contains proline in positions 2 and 3, and one ofeleven sequences contains no proline. The disclosed sequences arereferred to as REPLIKINES™. “REPLIKINE” is defined as a short sequencewithin ECM proteins that occurs multiple times (i.e., is replicated).This sequence may be present in one ECM protein (e.g., collagen IV).Preferably, the sequence is present in multiple ECM proteins (e.g., allcollagens, elastin, laminin, etc.). The presence of the sequence inmultiple ECM proteins increases the likelihood that the fragment may beable to promote ECM synthesis or repair.

The eleven GxxG sequences appearing in collagen IV listed above arehighlighted in the human collagen IV sequence illustrated in FIG. 1. Inthis figure, all bold sequences are underlined and overlapping sequencesare double-underlined. All but one of these sequences also appears incollagens I, II, III, and V. This fact contributes to the ability of thedisclosed peptides to stimulate the production of all collagen types,particularly when the peptides are used in combination. Table 1 showsthe frequency of several tetrapeptide repeats in ECM proteins. Boldsequences in Table I are those that appear in collagen IV ten or moretimes.

TABLE 1 Frequency of tetrapeptides in ECM proteins SEQ. CollagenCollagen Collagen Collagen Collagen Elastin ID NO Sequence I II III IV VElastin Precursor 19 GAAG 10 5 7 2 4 5 20 GAKG 3 4 3 5 5 21 GAPG 13 2125 6 9 22 GDKG 2 2 4 9 3 23 GDRG 2 5 2 4 1 8 GEKG 3 5 4 22 15 5 GEPG 1115 10 11 4 24 GERG 10 11 14 6 7 2 GFPG 4 8 6 22 5 1 1 25 GIPG 2 2 6 14 65 5 26 GKDG 1 4 5 2 2 27 GKPG 2 3 3 4 1 28 GLKG 2 1 1 5 4 29 GLPG 15 109 42 15 1 1 30 GNPG 3 5 3 2 1 31 GPAG 16 20 20 3 6 32 GPKG 3 11 4 12 9 7GPPG 33 40 40 46 43 33 GPQG 7 11 9 7 5 34 GPRG 11 13 10 4 7 35 GPSG 1011 5 1 5 36 GPTG 4 3 2 2 6 37 GPVG 9 3 3 2 5 38 GQPG 3 4 6 12 7 39 GRDG4 2 3 3 40 GRPG 3 3 4 2 5 3 GSPG 4 6 21 16 3 41 GTPG 3 4 2 11 2 42 GVKG1 3 2 3 1 43 GVPG 1 3 10 1 14 15 44 GYPG 1 1 1 4 2

As also evident from a review of the collagen IV sequence, SEQ ID NO:45,there are also many occurrences of sequences having the PxxP motif. Forexample, the sequence PGPP occurs no less than fifteen times asillustrated in FIG. 3. Therefore, this disclosed sequence is alsoreferred to as a REPLIKINE™. Preferably, this sequence is present inmultiple ECM proteins (e.g., all collagens, elastin, laminin, etc.) asthe presence of this sequence in multiple ECM proteins increases thelikelihood that the fragment may be able to promote ECM synthesis orrepair. The fifteen PGPP sequences appearing in collagen IV listed aboveare highlighted and underlined in the human collagen IV sequenceillustrated in FIG. 3.

Example 2 Identification of Frame Shift Actives

In addition to the relatively high proportion of collagen IVtetrapeptide repeat sequences with the motif GxxG, other tetrapeptidesequences occurring one amino acid frame shift away from a GxxG or PxxPtetrapeptide sequence have been identified. These sequences may repeator occur only once within an ECM protein and may be located one aminoacid position away from either a GxxG or PxxP tetrapeptide sequence asdescribed herein. These tetrapeptide sequences are referred to as frameshift actives. Such frame shift actives may accordingly contain either aG or a P in either the second or third position depending on thedirection of frame shift. It has been further recognized that frameshift actives may be combined with other tetrapeptide sequencesdisclosed in this application forming a combikine. An example of such acombikine is H06 and H15.

One example of a frame shift active is GAGP or H12 (SEQ ID NO:6). H12(GAGP) appears one residue (or frame) shift from the GxxG tetrapeptideGGAG in Collagen III (SEQ ID NO:46) as illustrated in FIG. 2. In thisfigure, all frame shift active sequences are bold and underlined and theGxxG sequences occurring one frame shift away are double-underlined.Furthermore, as shown in Table 5, this tetrapeptide (GAGP) achieves goodresults for collagen production at 48 hours. Another example is thesequence PGPR, which is H10 (SEQ ID NO:4) which occurs eleven times inCollagens I-IV. As it appears multiple times in an individual ECMprotein, this tetrapeptide would further be considered a REPLIKINE. FIG.2 (SEQ ID NO:46) illustrates several instances of this tetrapeptide witheach occurring one frame shift from the GxxG tetrapeptide GPRG. Thisparticular frame shift active appears in multiple ECM proteins andtherefore increases the likelihood that the fragment may be able topromote ECM synthesis or repair.

Example 3 Identification of Repeat Sequences that Stimulate CollagenProduction

Several sequences identified in Examples 1 and 2 were synthesized usingstandard peptide chemistry and assayed for the stimulation of collagenfrom dermal fibroblasts. The synthesized peptides were amidated at theC-terminus, which rendered the tetrapeptides less susceptible toprotease degradation and increased their solubility compared to the freeacid forms. Human dermal fibroblasts were incubated in 96-well plates at37° C. and 5% CO₂ for 24 and 48 hours in 150 μL complete cell culturemedia (Cascade Biologics, Portland, Oreg.; Cat. No. M-106-500),supplemented with Low Serum Growth Supplement (Cascade Biologics,Portland, Oreg.; Cat. No. S-003-10) containing sample peptides at afinal peptide concentration of 50 μg/mL. Each well was seeded with10,000 cells. Following the incubation, 100-μL medium samples wererecovered from each well and assayed for collagen production

The assays were performed by Tebu-bio Laboratories (France) using theSIRCOL™ Collagen Assay Kit (Biocolor Assays, UK) following themanufacturer's protocol. The SIRCOL™ Collagen Assay is a quantitativedye-binding method designed for the analysis of soluble collagensreleased into culture medium by mammalian cells during in vitro culture.The collagen of the tested samples binds to the anionic SIRCOL™ dye. Thecollagen-dye complexes precipitate out of solution and are pelleted bycentrifugation. The recovered collagen-dye pellet was dissolved in analkaline solution prior to absorbance measurements. Duplicatemeasurements were taken at the 24 and 48 hour times from two separatesamples. The four measurements for each sample were averaged. Theabsorbance of reagent blanks, collagen standards, and samples weremeasured at 560 nm. The reagent blank absorbance was subtracted from theabsorbance from each sample at 24 and 48 hours.

Two separate data sets were used to generate two collagen standardcalibration curves. The first calibration curve was generated forpurposes of calculating the quantity of collagen in samples H6(combination of SEQ ID NOs:1-4), H7-H14 (SEQ ID NOs:1-8, respectively)and H15 (combination of SEQ ID NOs:5-8). The second calibration curvewas generated for calculating the quantity of collagen in samples H16(SEQ ID NO:9), H21-23 (SEQ ID NOs:10-12, respectively), H25-26 (SEQ IDNOs:13-14, respectively), or H29-30 (SEQ ID NOs:15-16, respectively),H32 (SEQ ID NO:17), H33 (combination of SEQ ID NOs:9-12), H34(combination of SEQ ID NOs:11-14), H35 (combination of SEQ IDNOs:13-16), H36 (combination of SEQ ID NOs:1, 6, 5, 8), H37 (SEQ IDNO:17) and H38 (SEQ ID NO:8) from the absorbance measurements wascreated by plotting the Abs_(560nm) of the known collagen standardsversus the respective concentrations of the collagen standards (inmicrograms) each time a series of assays were performed. With respect toeach data set, the same calibration curve was used for samples taken atthe 24 and 48 hour times (Tables 2A and 2B). Accordingly, differentstandard curves were prepared immediately prior to performing eachseries of assays.

TABLE 2A Calibration curve for assaying collagen production by peptidesH6-H15 Collagen standards A_(560 nm) 24 h A_(560 nm) 48 h (μg) test test0 0.00 0.00 5 0.08 0.10 10 0.11 0.15 25 0.32 0.35 50 0.66 0.65

TABLE 2B Calibration curve for assaying collagen production by peptidesH16, H21-23, H25-26, and H29-38 Collagen Standards A_(560 nm) A_(560 nm)(μg) Assay date 1 Assay date 2 0 0.00 0.00 5 0.12 0.09 10 0.14 0.15 250.48 0.42 50 0.88 0.80

A linear regression was performed from plotting the Abs_(560nm) valuesversus concentrations of the respective collagen standards usingMICROSOFT EXCEL™. The regression resulted in a lines described by theformula y=0.013x for both incubation times noted in Table 2A. As theresults were identical, only the 24-hour time period was used for thesecond series calibration curves. The formula of the line obtained onassay date 1 and assay date 2 of the second series of samples wasy=0.0178x and y=0.0162x, respectively. The peptide LL-37 (SEQ ID NO:18)was used as a positive control as it has been widely reported to have animpact upon wound healing in man (Heilborn et al., The CathelicidinAnti-Microbial Peptide LL-37 Is Involved In The Re-Epithelialization OfHuman Skin Wounds And Is Lacking In Chronic Ulcer Epithelium, J. Invest.Dermato. 120: 379-89 (2003)). The assay detection limit defined by themanufacturer is 2.5 μg.

The total amount of collagen produced in samples containing peptides wascalculated from the averaged absorbance values taken at 24 hours (Table3A) and 48 hours (Table 3B) using the linear equation derived from thestandard curve. The total amount of collagen produced in samplescontaining peptides H16 (SEQ ID NO:9), H21-23 (SEQ ID NOs:10-12,respectively), H25-26 (SEQ ID NOs:13-14, respectively), or H29-30 (SEQID NOs:15-16, respectively), H32 (SEQ ID NO:17), H33 (combination of SEQID NOs:9-12), H34 (combination of SEQ ID NOs:11-14), H35 (combination ofSEQ ID NOs:13-16), H36 (combination of SEQ ID NOs:1, 6, 5, 8), H37 (SEQID NO:17) and H38 (SEQ ID NO:8) was calculated from the absorbancevalues taken at 24 hours (Table 4A) and 48 hours (Table 4B) using thelinear equation derived from the standard curve. These values werecompared with peptide LL37 (SEQ ID NO:18), a peptide known to stimulatecollagen. In each table, samples marked by an asterisk (*) may not besignificant as the assay detection limit is 2.5 μg.

TABLE 3A Absorbance measurements and quantification of collagen in testsamples H6-H15 at 24 hours. SEQ Average minus Collagen ID NO PeptidesA_(560 nm) Average blank (μg) 18  LL37 0.102 0.136 0.12 0.04 3.0 — H60.084 0.140 0.11 0.03 2.5 1 H7 0.098 0.063 0.08 0.00 0.0* 2 H8 0.1220.078 0.10 0.02 1.5* 3 H9 0.147 0.104 0.13 0.05 3.5 4 H10 0.103 0.1460.12 0.04 3.4 5 H11 0.110 0.168 0.14 0.06 4.5 6 H12 0.063 0.101 0.080.00 0.2* 7 H13 0.114 0.093 0.10 0.02 1.8* 8 H14 0.115 0.122 0.12 0.043.0 — H15 0.132 0.093 0.11 0.03 2.5 — Blank 0.074 0.076 0.08 0.00 0.0

TABLE 3B Absorbance measurements and quantification of collagen in testsamples H6-H15 at 48 hours. SEQ Average minus Collagen ID NO PeptidesA_(560 nm) Average blank (μg) 18  LL37 0.262 0.113 0.19 0.07 5.2 — H60.086 0.189 0.14 0.02 1.3* 1 H7 0.192 0.189 0.19 0.07 5.4 2 H8 0.1370.126 0.13 0.01 0.9* 3 H9 0.117 0.061 0.09 0.00 0.0* 4 H10 0.136 0.0850.11 0.00 0.0* 5 H11 0.113 0.181 0.15 0.03 2.1* 6 H12 0.106 0.231 0.170.05 3.7 7 H13 0.100 0.145 0.12 0.00 0.2* 8 H14 0.132 0.176 0.15 0.032.6 — H15 0.177 0.174 0.18 0.06 4.3 — Blank 0.120 0.115 0.12 0.00 0.0

TABLE 4A Absorbance measurements and quantification of collagen in testsamples H16, H21-23, H25-26, or H29-38 at 24 hours. SEQ ID AverageCollagen NO Peptides A_(560 nm) Average minus blank (μg)  9 H16 0.1330.137 0.14 0.06 3.1 10 H21 0.129 0.119 0.12 0.04 2.5 11 H22 0.192 0.0850.14 0.06 3.3 12 H23 0.090 0.073 0.08 0.00 0.1* 13 H25 0.129 0.076 0.100.02 1.3* 14 H26 0.114 0.149 0.13 0.05 2.9 15 H29 0.111 0.063 0.09 0.010.4* 16 H30 0.099 0.092 0.10 0.02 0.9* 17 H32 0.087 0.055 0.07 −0.01−0.5* (crystals and cell toxicity) — H33 0.086 0.125 0.11 0.03 1.4* —H34 0.117 0.120 0.12 0.04 2.2* — H35 0.103 0.090 0.10 0.02 0.9* — H360.105 0.128 0.12 0.04 2.1* 17 H37 0.099 0.100 0.10 0.02 1.1*  8 H380.103 0.159 0.13 0.05 2.9 — Blank 0.072 0.086 0.08 0.00 0.0

TABLE 4B Absorbance measurements and quantification of collagen in testsamples H16, H21-23, H25-26, or H29-38 at 48 hours. SEQ ID Average minusCollagen NO Peptides A_(560 nm) Average blank (μg)  9 H16 0.065 0.0640.06 0.00 0.3* 10 H21 0.089 0.126 0.11 0.05 2.9 11 H22 0.102 0.087 0.090.03 2.1* 12 H23 0.093 0.082 0.09 0.03 1.7* 13 H25 0.059 0.084 0.07 0.010.7* 14 H26 0.081 0.153 0.12 0.06 3.5 15 H29 0.086 0.094 0.09 0.03 1.9*16 H30 0.083 0.101 0.09 0.03 2.0* 17 H32 0.088 0.072 0.08 0.02 1.2*(crystals and cell toxicity) — H33 0.096 0.092 0.09 0.03 2.1* — H340.076 0.155 0.12 0.06 3.4 — H35 0.120 0.074 0.10 0.04 2.3* — H36 0.1540.082 0.12 0.06 3.6 17 H37 0.078 0.114 0.10 0.04 2.2*  8 H38 0.123 0.0890.11 0.05 2.8 — Blank 0.106 0.0106 0.06 0.00 0.0

Because sample sizes were 100 μL, the concentration of collagen producedin each sample in micrograms per milliliter is determined by multiplyingthe amount of collagen detected by ten. The results of all samplestested are summarized in Table 5.

TABLE 5 Collagen synthesis induced by peptides Collagen produced SEQ[Peptide] (μg/mL) ID NO Name Primary sequence (μg/mL). 24 hrs 48 hrs 1H07 PEGP 50 0 54 2 H08 GFPG 50 15 9 3 H09 GSPG 50 35 0 4 H10 PGPR 50 340 — H06 H7, H8, H9, H10 50 25 13 (SEQ ID NOs: 1, 2, 3, 4) 5 H11 GEPG 5045 21 6 H12 GAGP 50 2 37 7 H13 GPPG 50 18 2 8 H14 GEKG 50 30 26 8 H38GEKG 0.3 29 28 — H15 H11, H12, H13, H14 50 25 43 (SEQ ID NOs: 5, 6, 7,8) 9 H16 PEKP 50 31 3 10 H21 PKGP 50 25 29 11 H22 PGQP 50 33 21 12 H23PGTP 50 1 17 13 H25 PMGP 50 13 7 14 H26 PGPP 50 29 35 15 H29 PQGP 50 419 16 H30 PGNP 50 9 20 17 H32 KTTKS 50 na 12 (SEDERMA ™ peptide) 17 H37KTTKS 0.3 11 22 (SEDERMA ™ peptide) — H33 H16, H21, H22, H23 50 14 21(SEQ ID NOs: 9, 10, 11, 12) — H34 H22, H23, H25, H26 50 22 34 (SEQ IDNOs: 11, 12, 13, 14) — H35 H25, H26, H29, H30 50 9 23 (SEQ ID NOs: 13,14, 15, 16) — H36 H7, H12, H11, H14 50 21 36 (SEQ ID NOs: 1, 6, 5, 8) 18LL37 LLGDFFRKSKEKIGKEFKRIVQRIDFLR 50 30 52 NLVPRTES

All tetrapeptides tested stimulated the production of soluble collagen.Of the sequences tested, GxxG tetrapeptides with a glutamic acid inposition 2 best stimulate collagen at both 24 and 48 hour time-points.These sequences are H11 (GEPG; SEQ ID NO:5), H14 (GEKG; SEQ ID NO:8) andH38 (GEKG; SEQ ID NO:8). The peptides were initially screened using apeptide concentration of 50 jig/mL. To survey the concentrationeffective for stimulating collagen production, H14 (SEQ ID NO:8) wasalso tested at 0.3 μg/mL as H38. As shown in Table 5, H38-inducedcollagen stimulation was not diminished at the lower concentration,indicating that the maximal stimulating concentration of SEQ ID NO:8 isat or below 0.3 μg/mL.

To test its efficacy, SEQ ID NO:8 (H14 and H38) was compared to thepeptide, LL37, (SEQ ID NO:18) which is known to stimulate collagenproduction. Based on the amount of collagen released by fibroblasts inresponse to LL37, 25 μg/mL was considered a significant amount ofcollagen released due to contact with a tetrapeptide. SEQ ID NO:8induced about the same amount of collagen as LL37 (SEQ ID NO: 18) at 24hours. Importantly, collagen produced as a result of contact with SEQ IDNO:8 was substantially maintained for at least 48 hours. SEQ ID NO:8 wasalso compared to a leading skin care peptide known to stimulate collagenproduction, KTTKS (SEQ ID NO:17) (Katayama et. al., J. BIOL. CHEM. 288:9941-9944 (1983)). KTTKS is an ingredient in the product MATRIXYL™(SEDERMA SAS, France). SEQ ID NO:8 stimulated more collagen productionthan the KTTKS (SEQ ID NO:17) peptide (Table 5) at 24 and 48 hours.

Example 4 Identification of Peptide Combinations That SynergisticallyEnhance Collagen Stimulation—COMBIKINES

Heterogeneous populations of active tetrapeptides may stimulate collagenproduction at a higher level than homogenous samples of tetrapeptides.The components of the heterogeneous composition are called COMBIKINES™.COMBIKINES are a group of REPLIKINES combined to produce a greater orbroader effect upon one or more target cell types. The peptides H11 (SEQID NO:5), H12 (SEQ ID NO:6), H13 (SEQ ID NO:7), and H14 (SEQ ID NO:8)were combined to a final concentration of 50 μg/mL and assayed using thesame protocol as for the individual peptides. As expected, the resultobtained at the 24 hour time point equaled the mean of the individualinduction scores. The combination of peptides at 48 hours, however,induced collagen to a level of 43 μg/mL. Surprisingly, this amount wasfar in excess of the anticipated mean (21 μg/mL) of the four individualpeptides (see Table 5). Thus, specific combinations of peptides maystimulate collagen production to a greater degree than the individualpeptides at the same concentration. Further, tetrapeptides from avariety of ECM sources such as collagen, laminin, and elastin mayproduce enhanced induction of a variety of ECM proteins (see Tables 1and 5).

Example 5 Cost-effective COMBIKINE Manufacturing for EnhancingStimulation of Collagen Production

The high cost of peptide synthesis limits the feasibility of producingof heterogeneous compositions of bioactive peptides. The presentinvention greatly mitigates this limitation. Because the presentlydisclosed sequences have a commonality (e.g., a glycine or proline atboth termini), a range of tetrapeptides varied at positions 2 and 3 canbe synthesized in a single manufacturing run. The synthetic peptides canbe made by any method known in the art. (Benoiton, N., Chemistry ofPeptide Synthesis, CRC (2005)). During manufacture of the peptides,amino acid mixtures are added instead of homogenous samples. Thechemistry for determining the correct ratios of amino acidconcentrations added at the mixed positions to gain the desired ratio ofresulting peptides has been described previously (Greenbaum et al.,Molecular and Cellular Proteomics 1: 60-68, 2002; Krstenansky et al.,Letters in Drug Design and Discovery 1: 6-13, 2004; both of whichreferences are incorporated herein in their entirety). Using thismethodology, a library of heterogeneous peptides can be made for nearlythe same cost of synthesizing one peptide.

The application of this manufacturing process enables the cost-effectiveproduction of bioactive combikines. This is made possible by the uniquecomposition of the disclosed tetrapeptides. The tetrapeptide mixturesare better suited for incorporation into topical use formulations thanlonger peptides. Because of their length, tetrapeptides have practicaland chemical advantages over longer peptides, including the following:easier incorporation and dissolution into formulations, higher skin andpore permeability, and higher production yields with easier methods ofmanufacturing combinations of peptides. Although not required, the idealformulations of tetrapeptides, singly or in combination, areformulations that maintain significant collagen production at 24 hoursfor up to 48 hours. More preferably, the formulations would inducesynthesis of ECM for the entire 48 hour period such that more collagenis produced by 48 hours than at 24 hours. Although within the scope ofthe current invention, tetrapeptides that promote production of ECMproteins at 24 hours, but show diminished production at 48 hours, areless favored. In this regard, Table 6 shows the results of the currentlydisclosed peptides. Preferred peptides are in bold.

TABLE 6 Disclosed peptides Released Released Significant IncreaseDecrease collagen collagen release in collagen in collagen SEQ ID(μg/mL) (μg/mL) of collagen release release NO Peptides 24 h 48 h at 24h and 48 h at 48 h v. 24 h at 48 h v. 24 h 18 LL37 30 52 ✓ ✓ — H6 25 13 1 H7 0 54 ✓  2 H8 15 9  3 H9 35 0 ✓  4 H10 34 0 ✓  5 H11 45 21 ✓  6 H122 37 ✓  7 H13 18 2  8 H14 30 26 ✓  8 H38 29 28 ✓ — H15 25 43 ✓ ✓  9 H1631 3 ✓ 10 H21 25 29 ✓ 11 H22 33 21 ✓ 12 H23 1 17 ✓ 13 H25 13 7 ✓ 14 H2629 35 ✓ 15 H29 4 19 ✓ 16 H30 9 20 ✓ 17 H32 NA 12 (crystals and celltoxicity) 17 H37 11 22 ✓ — H33 14 21 ✓ — H34 22 34 ✓ — H35 9 23 ✓ — H3621 36 ✓

Example 6 Collagen Stimulators Also Serve as Multi-effector MoleculesEnhancing Skin Epithelial Cell Wound Closer

Collagens are key components of all phases of wound healing. Stimulationof collagen production reflects that damage has occurred to the collagennetwork (e.g. by enzymes or physical destruction). Indeed, the totalcollapse of the collagen network in fact causes healing to take place.Therefore a collagen stimulator may also serve as a multi-effectormolecule orchestrating certain matrix remodeling and enhancing woundhealing.

Wound healing experiments were performed on monolayers of human skinepithelial cells (CRL-2592) plated onto 12-well plates. Cells wereserum-starved for 24 hours before experimentation. Confluent monolayersof CRL-2592 were wounded using a P200 (200-EL) pipette tip. The woundswere washed and picture-documented prior to peptide treatment. Peptideswere added to a final concentration from 20 to 40 μg/ml. Cells were keptin an incubator at 37° C., 5% CO₂, and 92% humidity, except when imageswere being captured for a short period at room temperature. Woundclosure was followed at 6-hour and 10-hour time points. PBS-treatedwounds were used as negative controls for comparison purposes.

TABLE 7 Effect of peptides on human skin epithelial wound closure invitro 0 hr 6 hr 10 hr Compound W-size* W-size % closure W-size % closurePBS-1 36 29 19.40% 21 41.70% PBS-2 52 42 19.20% 30 42.30% SEQ ID NO: 1425 12   52% 2.75   89% SEQ ID NO: 5 48 39   19% 30 37.50% *W-size: woundsize (arbitrary)

In vitro monolayer wound closure is a result of cell migration, which isimportant in many biological processes such as embryogenesis,angiogenesis, inflammatory reactions and wound repair. These processesare thought to be regulated by interactions with other cells, cytokinesand ECM proteins. As shown in Table 7, SEQ ID NO:14 significantlyinduces wound closure compared to the effects of PBS alone. Suchactivity is peptide-specific as well as cell type-specific since SEQ IDNO:14 does not induce wound closure in a human skin fibroblast monolayer(data not shown). SEQ ID NO:5 is also a collagen inducer, but does notenhance wound closure or epithelial cell migration to any great extentcompared to the effects of PBS alone. The fact that SEQ ID NO:14 inducedcell migration or wound closure in a manner specific to skin epithelialcells (i.e. does not recruit fibroblasts) may add an advantage to usingthis peptide for skin care, since it is believed that the recruitment oflarge numbers of active fibroblasts to a wound site results in excessdeposition and contraction of tissue resulting in scarring.

All of the compositions or methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and/or methods and in the steps or in the sequence of stepsof the methods described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention.

1. A tetrapeptide capable of inducing production of an extracellular matrix protein, wherein the tetrapeptide comprises SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:40, or SEQ ID NO:41.
 2. The tetrapeptide of claim 1, wherein the tetrapeptide comprises SEQ ID NO:5.
 3. The tetrapeptide of claim 2, wherein the tetrapeptide is SEQ ID NO:5.
 4. The tetrapeptide of claim 1, wherein the tetrapeptide comprises SEQ ID NO:3.
 5. The tetrapeptide of claim 4, wherein the tetrapeptide is SEQ ID NO:3.
 6. The tetrapeptide of claim 1, wherein the tetrapeptide is amidated at the carboxy-terminus.
 7. The tetrapeptide of claim 1, wherein the extracellular matrix protein is collagen.
 8. A composition comprising at least one tetrapeptide of claim 1 and a pharmaceutically acceptable carrier.
 9. The composition of claim 8, wherein the tetrapeptide is present in a concentration ranging from about 0.01 μg/mL to about 100 μg/mL.
 10. The composition of claim 8, wherein the tetrapeptide is present in a concentration ranging from about 0.1 μg/mL to about 1 μg/mL.
 11. The composition of claim 8, wherein the composition is in the form of an aerosol, emulsion, liquid, lotion, cream, paste, ointment, or foam.
 12. A method for stimulating the production of collagen in a human, the method comprising administering to said human a therapeutically effective amount of the composition of claim
 8. 13. The method of claim 12, wherein the therapeutically effective amount of the composition comprises about 0.1 μg/mL to about 50 μg/mL of tetrapeptide.
 14. The method of claim 12, wherein the administering step comprises topically applying the composition to damaged skin.
 15. A tetrapeptide capable of inducing production of an extracellular matrix protein comprising the amino acid sequence PGPR (SEQ ID NO:4).
 16. The tetrapeptide of claim 1, wherein the tetrapeptide comprises SEQ ID NO:8.
 17. The tetrapeptide of claim 16, wherein the tetrapeptide is SEQ ID NO:8.
 18. The tetrapeptide of claim 1, wherein the tetrapeptide comprises SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:30, SEQ ID NO:40, or SEQ ID NO:41.
 19. The composition of claim 8, where said composition comprises the tetrapeptide of SEQ ID NO:5.
 20. The method of claim 14, wherein said damaged skin is a result of aging, disease, injury, trauma, or surgery.
 21. The method of claim 14, wherein the composition comprises a tetrapeptide in combination with a pharmaceutically acceptable carrier, and wherein said tetrapeptide comprises SEQ ID NO:5.
 22. The method of claim 21, wherein the composition further comprises the tetrapeptides comprising SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.
 23. The method of claim 14, wherein the composition comprises a tetrapeptide in combination with a pharmaceutically acceptable carrier, and wherein said tetrapeptide comprises SEQ ID NO:8.
 24. The composition of claim 19, wherein said composition further comprises the tetrapeptides comprising SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. 